A. Methods for Monitoring Autophagy

1. 
   Transmission electron microscopy. Autophagy was first detected by TEM in the 1950s (reviewed in ref. ⁵). It was originally observed as focal degradation of cytoplasmic areas performed by lysosomes, which remains the hallmark of this process. Later analysis revealed that it starts with the sequestration of portions of the cytoplasm by a special double membrane structure (now termed the phagophore), which matures into the autophagosome, still bordered by a double membrane. Subsequent fusion events transport the cargo to the lysosome (or the vacuole in fungi or plants) for enzymatic breakdown.
   

Autophagy can be both selective and nonselective, and TEM can be used to monitor both. In the case of selective autophagy, the cargo is the specific substrate being targeted for sequestration—bulk cytoplasm is essentially excluded. In contrast, during nonselective autophagy, the various cytoplasmic constituents are sequestered randomly, resulting in autophagosomes in the size range of normal mitochondria. Sequestration of larger structures (such as big lipid droplets, extremely elongated or branching mitochondria or the entire Golgi complex) is rare, indicating an apparent upper size limit for individual autophagosomes. However, it has been observed that under special circumstances the potential exists for the formation of huge autophagosomes, which can even engulf a complete nucleus.²⁴ Cellular components that form large confluent areas excluding bulk cytoplasm, such as glycogen or organized, functional myofibrillar structures, do not seem to be sequestered by macroautophagy.

After sequestration, the content of the autophagosome and its bordering double membrane remain morphologically unchanged, and clearly recognizable for a considerable time, which can be measured for at least many minutes. During this period, the membranes of the sequestered organelles (for example the ER or mitochondria) remain intact, and the density of ribosomes is conserved at normal levels. Degradation and the sequestered material and the corresponding deterioration of ultrastructure commences and runs to completion within the amphisome and the autolysosome after fusion with a late endosome and lysosome (the vacuole in fungi and plants), respectively (Fig. 1).⁴⁴ The sequential morphological changes during the autophagic process can be followed by TEM. The maturation from the phagophore through the autolysosome is a dynamic and continuous process,⁴⁵ and, thus, the classification of compartments into discrete morphological subsets can be problematic; therefore, some basic guidelines are offered below.

In the preceeding sections the “autophagosome”, the “amphisome” and the “autolysosome” were terms used to describe or indicate 3 basic stages and compartments of autophagy. It is important to make it clear that for instances (which may be many) when we cannot or do not want to differentiate among the autophagosomal, amphisomal and autolysosomal stage we use the general term “autophagic vacuole”. In the yeast autophagy field the term “autophagic vesicle” is used to avoid confusion with the primary vacuole, and by now the 2 terms are used in parallel and can be considered synonyms. It is strongly recommended, however, to use only the term “autophagic vacuole” when referring to macroautophagy in higher eukaryotic cells. Autophagosomes, also referred to as initial autophagic vacuoles (AVi), typically have a double membrane. This structure is usually distinctly visible by EM as 2 parallel membrane layers (bilayers) separated by a relatively narrower or wider electron-translucent cleft, even when applying the simplest routine EM fixation procedure (Fig. 3A).^46,47 In the case of nonselective autophagy, autophagosomes contain cytosol and/or organelles appearing morphologically intact as also described above.^44,48 Amphisomes⁴⁹ can sometimes be identified by the presence of small internal vesicles within the autophagosome/autophagic vacuole (AV).⁵⁰ These internal vesicles are delivered into the lumen by fusion of the autophagosome/AV limiting membrane with multivesicular endo­somes, and care should therefore be taken in the identification of the organelles, especially in cells that produce large numbers of multivesicular body (MVB)-derived exosomes (such as tumor or stem cells).⁵¹ Late/degradative autophagic vacuoles/autolysosomes (AVd or AVl) typically have only one limiting membrane; frequently they contain electron dense cytoplasmic material and/or organelles at various stages of degradation (Fig. 3A and B);^44,48 although late in the digestion process, they may contain only a few membrane fragments and be difficult to distinguish from lysosomes, endosomes, or tubular smooth ER cut in cross-section. Unequivocal identification of these structures and of lysosomes devoid of visible content requires immuno-EM detection of a cathepsin or other lysosomal hydrolase (e.g., ACP2 [acid phosphatase 2, lysosomal]⁵²) that is detected on the limiting membrane of the lysosome.⁵³ Smaller, often electron dense, lysosomes may predominate in some cells and exhibit hydrolase immunoreactivity within the lumen and on the limiting membrane.⁵⁴

The presence of lytic enzymes in autolysosomes is an important criterion for identification, although, by itself, insufficient to distinguish them from late endosomes/MVBs, amphisomes, lysosomes and lysosomal-related residual bodies (e.g., lipofuscin), which also contain these enzyes. Traditional methods of detection involve demonstrating the activity of ACP2/acid phosphatase by enzyme cytochemistry⁵⁵ or showing the presence of the hydrolase by immunocytochemistry.⁵⁶ In addition, structural proteins of the lysosome/late endosome, such as LAMP1 and LAMP2 or SCARB2/LIMP-2, can be used for confirmation. No single protein marker, however, has been effective in discriminating autolysosomes from the compartments mentioned above, in part due to the dynamic fusion and “kiss-and-run” events that promote interchange of components that can occur between these organelle subtypes. Rigorous further discrimination of these compartments from each other and other vesicles ultimately requires demonstrating the colocalization of a second marker indicating the presence of an autophagic substrate (e.g., LC3-CTSD colocalization) or the acidification of the compartment (e.g., mRFP/mCherry-GFP-LC3 probes (see Tandem mRFP/mCherry-GFP fluorescence microscopy), or Bodipy-pepstatin A detection of CTSD in an activated form within an acidic compartment), and, when appropriate, by excluding markers of other vesicular components.^52,57,58

The sequential deterioration of cytoplasmic structures being digested can be used for identifying autolysosomes by TEM. Even when the partially digested and destroyed structure cannot be recognized in itself, it can be traced back to earlier forms by identifying preceeding stages of sequential morphological deterioration. Degradation usually leads first to increased density of still recognizable organelles, then to vacuoles with heterogenous density, which become more homogenous and amorphous, mostly electron dense, but sometimes light (i.e., electron translucent). It should be noted that, in pathological states, it is not uncommon that active autophagy of autolysosomes and damaged lysosomes (“lysosophagy”) may yield populations of double-memebrane limited autophagosomes containing partially digested amorphous substrate in the lumen either free or within smaller electron-dense vesicles. These structures, which are enriched in hydrolases, are frequently seen in swollen dystrophic neurites in some neurodegenerative diseases.⁵⁴

It must be emphasized that in addition to the autophagic input, other processes (e.g., endosomal, phagosomal, chaperone-mediated) also carry cargo to the lysosomes,^59,60 in some cases through the intermediate step of direct endosome fusion with an autophagosome to form an amphisome. This process is exceptionally common in the axons of neurons.⁶¹ Therefore, strictly speaking, we can only have a lytic compartment containing cargos arriving from several possible sources; however, we still may use the term “autolysosome” if the content appears to be overwhelmingly autophagic. Note that the engulfment of apoptotic cells via phagocytosis also produces lysosomes that contain cytoplasmic structures, but in this case it originates from the dying cell; hence the possibility of an extracellular origin for such content must be considered when monitoring autophagy in settings where apoptotic cell death may be reasonably expected or anticipated.

For many biological and pathological situations, examination of both early and late autophagic vacuoles yields valuable data regarding the overall autophagy status in the cells.¹⁴ Along these lines, it is possible to use immunocytochemistry to follow particular cytosolic proteins such as SOD1/CuZn superoxide dismutase and CA/carbonic anhydrase to determine the stage of autophagy; the former is much more resistant to lysosomal degradation.⁶² In some autophagy-inducing conditions it is possible to observe multi-lamellar membrane structures in addition to the conventional double-membrane autophagosomes, although the nature of these structures is not fully understood. These multi-lamellar structures may indeed be multiple double layers of phagophores⁶³ and positive for LC3,⁶⁴ they could be autolysosomes,⁶⁵ or they may form artifactually during fixation.

Special features of the autophagic process may be clarified by immuno-TEM with gold-labeling,^66,67 using antibodies, for example, to cargo proteins of cytoplasmic origin and to LC3 to verify the autophagic nature of the compartment. LC3 immunogold labeling also makes it possible to detect novel degradative organelles within autophagy compartments. This is the case with the autophagoproteasome where costaining for LC3 and ubiquitin-proteasome system (UPS) antigens occurs. The autophagoproteasome consists of single-, double-, or multiple-membrane LC3-positive autophagosomes costaining for specific components of the UPS. It may be that a rich multi-enzymatic (both autophagic and UPS) activity takes place within these organelles instead of being segregated within different cell domains.

Although labeling of LC3 can be difficult, an increasing number of commercial antibodies are becoming available, among them good ones to visualize the GFP moiety of GFP-LC3 reporter constructs.⁶⁸ It is important to keep in mind that LC3 can be associated with nonautophagic structures (see Xenophagy, and Noncanonical use of autophagy-related proteins). LC3 is involved in specialized forms of endocytosis like LC3-associated phagocytosis. In addition, LC3 can decorate vesicles dedicated to exocytosis in nonconventional secretion systems (reviewed in ref. ⁶⁹). Antibodies against an abundant cytosolic protein will result in high labeling all over the cytoplasm; however, organelle markers work well. Because there are very few characterized proteins that remain associated with the completed autophagosome, the choices for confirmation of its autophagic nature are limited. Furthermore, autophagosome-associated proteins may be cell type-specific. At any rate, the success of this methodology depends on the quality of the antibodies and also on the TEM preparation and fixation procedures utilized. With immuno-TEM, authors should provide controls showing that labeling is specific. This may require a quantitative comparisons of labeling over different cellular compartments not expected to contain antigen and those containing the antigen of interest.

In clinical situations it is difficult to demonstrate autophagy clearly in tissues of formalin-fixed and paraffin-embedded biopsy samples retrospectively, because (1) tissues fixed in formalin have low or no LC3 detectable by routine immunostaining, because phospholipids melt together with paraffin during the sample preparation, and (2) immunogold electron microscopy of many tissues not optimally fixed for this purpose (e.g., using rapid fixation) produces low-quality images. Combining antigen retrieval with the avidin-biotin peroxidase complex (ABC) method may be quite useful for these situations. For example, immunohistochemistry can be performed using an antigen retrieval method, then tissues are stained by the ABC technique using a labeled anti-human LC3 antibody. After imaging by light microscopy, the same prepared slides can be remade into sections for TEM examination, which can reveal peroxidase reaction deposits in vacuoles within the region that is LC3-immunopositive by light microscopy.⁷⁰

In addition, statistical information should be provided due to the necessity of showing only a selective number of sections. Again, we note that for quantitative data it is necessary to use proper volumetric analysis rather than just counting numbers of sectioned objects. On the one hand, it must be kept in mind that even volumetric morphometry/stereology only shows either steady state levels, or a snapshot in a changing dynamic process. Such data by themselves are not informative regarding autophagic flux, unless carried out over multiple time points. Alternatively, investigation in the presence and absence of flux inhibitors can reveal the dynamic changes in various stages of the autophagic process.^11,20,71,72^,41 On the one hand, if the turnover of autolysosomes is very rapid, a low number/volume will not necessarily be an accurate reflection of low autophagic activity. However, quantitative analyses indicate that autophagosome volume in many cases does correlate with the rates of protein degradation.^73-75 One potential compromise is to perform whole cell quantification of autophagosomes using fluorescence methods, with qualitative verification by TEM,⁷⁶ to show that the changes in fluorescent puncta reflect corresponding changes in autophagic structures.

One additional caveat with TEM, and to some extent with confocal fluorescence microscopy, is that the analysis of a single plane within a cell can be misleading and may make the identification of autophagic structures difficult. Confocal microscopy and fluorescence microscopy with deconvolution software (or with much more work, 3-dimensional TEM) can be used to generate multiple/serial sections of the same cell to reduce this concern; however, in many cases where there is sufficient structural resolution, analysis of a single plane in a relatively large cell population can suffice given practical limitations. Newer EM technologies, including focused ion beam dual-beam EM, should make it much easier to apply three-dimensional analyses. An additional methodology to assess autophagosome accumulation is correlative light and electron microscopy (CLEM), which is helpful in confirming that fluorescent structures are autophagosomes.^77-79 Along these lines, it is important to note that even though GFP fluorescence will be quenched in the acidic environment of the autolysosome, some of the GFP puncta detected by light microscopy may correspond to early autolysosomes prior to GFP quenching. The mini Singlet Oxygen Generator (miniSOG) fluorescent flavoprotein, which is less than half the size of GFP, provides an additional means to genetically tag proteins for CLEM analysis under conditions that are particularly suited to subsequent TEM analysis.⁸⁰ Combinatorial assays using tandem monomeric red fluorescent protein (mRFP)-GFP-LC3 (see Tandem mRFP/mCherry-GFP fluorescence microscopy) along with static TEM images should help in the analysis of flux and the visualization of cargo structures.⁸¹

Another technique that has proven quite useful for analyzing the complex membrane structures that participate in autophagy is three-dimensional electron tomography,^82,83 and cryoelectron microscopy (Fig. 4). More sophisticated, cryo-soft X-ray tomography (cryo-SXT) is an emerging imaging technique used to visualize autophagosomes.⁸⁴ Cryo-SXT extracts ultrastructural information from whole, unstained mammalian cells as close to the “near- native” fully-hydrated (living) state as possible. Correlative studies combining cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) have been applied to capture early autophagosomes.

Finally, although only as an indirect measurement, the comparison of the ratio of autophagosomes to autolysosomes by TEM can support alterations in autophagy identified by other procedures.⁸⁵ In this case it is important to always compare samples to the control of the same cell type and in the same growth phase, as the autophagosome/autolysosome ratio varies in a cell context-dependent fashion, depending on their clearance activity. It may also be necessary to distinguish autolysosomes from telolysosomes/late secondary lysosomes (the former are actively engaged in degradation, whereas the latter have reached an end point in the breakdown of lumenal contents) because lysosome numbers generally increase when autophagy is induced. An additional category of lysosomal compartments, especially common in disesase states and aged postmitotic cells such as neurons is the residual body. This category includes ceroid and lipofuscin, lobulated vesicular compartments of varying size composed of highly indigestible complexes of protein and lipid and abundant, mostly inactive, acid hydrolases. Reflecting end-stage unsuccessful incomplete autolysosomal digestion, lipofuscin is fairly easily distinguished from AVs and lysosomes by TEM but can be easily confused with autolysosomes in immunocytochemistry studies at the light microscopy level.⁵²

TEM observations of platinum-carbon replicas obtained by the freeze fracture technique can also supply useful ultrastructural information on the autophagic process. In quickly frozen and fractured cells the fracture runs preferentially along the hydrophobic plane of the membranes, allowing characterization of the limiting membranes of the different types of autophagic vacuoles and visualization of their limited protein intramembrane particles (IMPs, or integral membrane proteins). Several studies have been carried out using this technique on yeast,⁸⁶ as well as on mammalian cells or tissue; first on mouse exocrine pancreas,⁸⁷ then on mouse and rat liver,^88,89 mouse seminal vesicle epithelium,^24,63 rat tumor and heart,⁹⁰ or cancer cell lines (e.g., breast cancer MDA-MB-231)⁹¹ to investigate the various phases of autophagosome maturation, and to reveal useful details about the origin and evolution of their limiting membranes.^5,92-95

The phagophore and the limiting membranes of autophagosomes contain few, or no detectable, IMPs (Fig. 5A,B), when compared to other cellular membranes and to the membranes of lysosomes. In subsequent stages of the autophagic process the fusion of the autophagosome with an endosome and a lysosome results in increased density of IMPs in the membrane of the formed autophagic compartments (amphisomes, autolysosomes; Fig. 5C).^{5,24,86-89,96,97} Autolysosomes are delimited by a single membrane because, in addition to the engulfed material, the inner membrane is also degraded by the lytic enzymes. Similarly, the limiting membrane of autophagic bodies in yeast (and presumably plants) is also quickly broken down under normal conditions. Autophagic bodies can be stabilized, however, by the addition of phenylmethylsulphonylfluoride (PMSF) or genetically by the deletion of the yeast PEP4 gene (see The Cvt pathway, mitophagy, pexophagy, piecemeal microautophagy of the nucleus and late nucleophagy in yeast and filamentous fungi.). Thus, another method to consider for monitoring autophagy in yeast (and potentially in plants) is to count autophagic bodies by TEM using at least 2 time points. The advantage of this approach is that it can provide accurate information on flux even when the autophagosomes are abnormally small.^98,99 Thus, although a high frequency of “abnormal” structures presents a challenge, TEM is still very helpful in analyzing autophagy.

Cautionary notes: Despite the introduction of many new methods TEM maintains its special role in autophagy research. There are, however, difficulties in utilizing TEM. It is relatively time consuming, and needs technical expertise to ensure proper handling of samples in all stages of preparation from fixation to sectioning and staining (contrasting). After all these criteria are met, we face the most important problem of proper identification of autophagic structures. This is crucial for both qualitative and quantitative characterization, and needs considerable experience, even in the case of one cell type. The difficulty lies in the fact that many subcellular components may be mistaken for autophagic structures. For example, some authors (or reviewers of manuscripts) assume that almost all cytoplasmic structures that, in the section plane, are surrounded by 2 (more or less) parallel membranes are autophagosomes. Structures appearing to be limited by a double membrane, however, may include swollen mitochondria, plastids in plant cells, cellular interdigitations, endocytosed apoptotic bodies, circular structures of lamellar smooth endoplasmic reticulum (ER), and even areas surrounded by rough ER. Endosomes, phagosomes and secretory vacuoles may have heterogenous content that makes it possible to confuse them with autolysosomes. Additional identification problems may arise from damage caused by improper sample taking or fixation artifacts.^{46,47,100,101}

Whereas fixation of in vitro samples is relatively straightforward, fixation of excised tissues requires care to avoid sampling a nonrepresentative, uninformative, or damaged part of the tissue. For instance, if 95% of a tumor is necrotic, TEM analysis of the necrotic core may not be informative, and if the sampling is from the viable rim, this needs to be specified when reported. Clearly this introduces the potential for subjectivity because reviewers of a paper cannot request multiple images with a careful statistical analysis with these types of samples. In addition, ex vivo samples are not typically randomized during processing, further complicating the possibility of valid statistical analyses. Ex vivo tissue should be fixed immediately and systematically across samples to avoid changes in autophagy that may occur simply due to the elapsed time ex vivo. It is recommended that for tissue samples, perfusion fixation should be used when possible. For yeast, rapid freezing techniques such as high pressure freezing followed by freeze substitution (i.e., dehydration at low temperature) may be particularly useful.

Quantification of autophagy by TEM morphometry has been rather controversial, and unreliable procedures still continue to be used. For the principles of reliable quantification and to avoid misleading results, excellent reviews are available.^10,102-104 In line with the basic principles of morphometry we find it necessary to emphasize here some common problems with regard to quantification. Counting autophagic vacuole profiles in sections of cells gives totally unreliable results, partly because both cell areas and profile areas are variable and also because the frequency of section profiles depends on the size of the vacuoles. There are morphometric procedures to measure or estimate the size range and the number of spherical objects by profiles in sections;¹⁰³ however, such methods have been used in autophagy research only a few times.^{31,99,105,106}

Proper morphometry described in the cited reviews will give us data expressed in µm³ autophagic vacuole/µm³ cytoplasm for relative volume (also called volume fraction or volume density), or µm² autophagic vacuole surface/µm³ cytoplasm for relative surface (surface density). Examples of actual morphometric measurements for the characterization of autophagic processes can be found in several articles.^{20,100,103,107,108} It is appropriate to note here that a change in the volume fraction of the autophagic compartment may come from 2 sources; from the real growth of its size in a given cytoplasmic volume, or from the decrease of the cytoplasmic volume itself. To avoid this so-called “reference trap,” the reference space volume can be determined by different methods.^104,109 If different magnifications are used for measuring the autophagic vacuoles and the cytoplasm (which may be practical when autophagy is less intense) correction factors should always be used.

In some cases, it may be prudent to employ tomographic reconstructions of the TEM images to confirm that the autophagic compartments are spherical and are not being confused with interdigitations observed between neighboring cells, endomembrane cisternae or damaged mitochondria with similar appearance in thin-sections (e.g., see ref. ¹¹⁰), but this is obviously a time-consuming approach requiring sophisticated equipment. In addition, interpretation of tomographic images can be problematic. For example, starvation-induced autophagosomes should contain cytoplasm (i.e., cytosol and possibly organelles), but autophagosome-related structures involved in specific types of autophagy should show the selective cytoplasmic target, but may be relatively devoid of bulk cytoplasm. Such processes include selective peroxisome or mitochondria degradation (pexophagy or mitophagy, respectively),^111,112 targeted degradation of pathogenic microbes (xenophagy),^113-118 a combination of xenophagy and stress-induced mitophagy,¹¹⁹ as well as the yeast biosynthetic cytoplasm-to-vacuole targeting (Cvt) pathway.¹²⁰ Furthermore, some pathogenic microbes express membrane-disrupting factors during infection (e.g., phospholipases) that disrupt the normal double-membrane architecture of autophagosomes.¹²¹ It is not even clear if the sequestering compartments used for specific organelle degradation or xenophagy should be termed autophagosomes or if alternate terms such as pexophagosome,¹²² mitophagosome and xenophagosome should be used, even though the membrane and mechanisms involved in their formation may be identical to those for starvation-induced autophagosomes; for example, the double-membrane vesicle of the Cvt pathway is referred to as a Cvt vesicle.¹²³

The confusion of heterophagic structures with autophagic ones is a major source of misinterpretation. A prominent example of this is related to apoptosis. Apoptotic bodies from neighboring cells are readily phagocytosed by surviving cells of the same tissue.^124,125 Immediately after phagocytic uptake of apoptotic bodies, phagosomes may have double limiting membranes. The inner one is the plasma membrane of the apoptotic body and the outer one is that of the phagocytizing cell. The early heterophagic vacuole formed in this way may appear similar to an autophagosome or, in a later stage, an early autolysosome in that it contains recognizable or identifiable cytoplasmic material. A major difference, however, is that the surrounding membranes are the thicker plasma membrane type, rather than the thinner sequestration membrane type (9-10 nm, versus 7-8 nm, respectively).¹⁰¹ A good feature to distinguish between autophagosomes and double plasma membrane-bound structures is the lack of the distended empty space (characteristic for the sequestration membranes of autophagosomes) between the 2 membranes of the phagocytic vacuoles. In addition, engulfed apoptotic bodies usually have a larger average size than autophagosomes.^126,127 The problem of heterophagic elements interfering with the identification of autophagic ones is most prominent in cell types with particularly intense heterophagic activity (such as macrophages, and amoeboid or ciliate protists). Special attention has to be paid to this problem in cell cultures or in vivo treatments (e.g., with toxic or chemotherapeutic agents) causing extensive apoptosis.

The most common organelles confused with autophagic vacuoles are mitochondria, ER, endosomes, and also (depending on their structure) plastids in plants. Due to the cisternal structure of the ER, double membrane-like structures surrounding mitochondria or other organelles are often observed after sectioning,¹²⁸ but these can also correspond to cisternae of the ER coming into and out of the section plane.⁴⁶ If there are ribosomes associated with these membranes they can help in distinguishing them from the ribosome-free double-membrane of the phagophore and autophagosome. Observation of a mixture of early and late autophagic vacuoles that is modulated by the time point of collection and/or brief pulses of bafilomycin A₁ (a vacuolar-type H⁺-ATPase [V-ATPase] inhibitor) to trap the cargo in a recognizable early state⁴¹ increases the confidence that an autophagic process is being observed. In these cases, however, the possibility that feedback activation of sequestration gets involved in the autophagic process has to be carefully considered. To minimize the impact of errors, exact categorization of autophagic elements should be applied. Efforts should be made to clarify the nature of questionable structures by extensive preliminary comparison in many test areas. Elements that still remain questionable should be categorized into special groups and measured separately. Should their later identification become possible, they can be added to the proper category or, if not, kept separate.

For nonspecialists it can be particularly difficult to distinguish among amphisomes, autolysosomes and lysosomes, which are all single-membrane compartments containing more or less degraded material. Therefore, we suggest in general to measure autophagosomes as a separate category for a start, and to compile another category of degradative compartments (including amphisomes, autolysosomes and lysosomes). All of these compartments increase in quantity upon true autophagy induction; however, in pathological states, it may be informative to discriminate among these different forms of degradative compartments, which may be differentially affected by disease factors.

In yeast, it is convenient to identify autophagic bodies that reside within the vacuole lumen, and to quantify them as an alternative to the direct examination of autophagosomes. However, it is important to keep in mind that it may not be possible to distinguish between autophagic bodies that are derived from the fusion of autophagosomes with the vacuole, and the single-membrane vesicles that are generated during microautophagy-like processes such as micropexophagy and micromitophagy.